I= AN INVESTIGATION OF ANTIMICROBIAL COMPOUNDS FOR IMMUNOMODULATING AND ANTI-ADHESION PROPERTIES
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Title of Thesis
AN INVESTIGATION OF ANTIMICROBIAL COMPOUNDS FOR IMMUNOMODULATING AND ANTI-ADHESION PROPERTIES

Author(s)
Nafisa Hassan Ali
Institute/University/Department Details
Department of Microbiology/ University of Karachi
Session
2003
Subject
Microbiology
Number of Pages
121
Keywords (Extracted from title, table of contents and abstract of thesis)
antimicrobial compounds, immunomodulating, anti-adhesion properties, antimicrobial products, cell mediated immune responses, cariogenic bacteria, uropathogenic e. Coli, s. Agalactiae vaginal infection

Abstract
Global emergence of resistance in bacteria to the antimicrobial agents has made it difficult for the physicians to find effective therapeutic agents for diseases caused by MDR - TB, MDR-Salmonella, MRSA, Ps. aeruginosa, Acinetobacter, Candida etc. Since long infectious disease are being cured by traditional practitioners with the help of herbs and higher plants. At present antibiotics, obtained from molds fungus or synthetically prepared have lost their efficacy. The new trend to combat the pathogens is to use water and organic extracts made from different parts of medicinal plants.

In this study 30 medicinal plants and their different parts were screened for their in-vitro anti- microbial activity, anti-adhesion activity under in -vitro and in- vivo in rat model, their toxicity and immunomodulating properties in BALB/c mice.

Screening for the antimicrobial activity was carried out against twenty different bacterial species that includes A TCC and clinical strains. The screening of antibacterial activity was performed by the agar well diffusion and micro broth dilution method. Beside water solution of Rosa centrifolia (rose) and fruit, leaves and stem solutions of C. fistula was also included. From among eleven plant spices, only clove aqueous extract showed broad-spectrum of antibacterial activity against Ps. Aerugenosae and S. agalactiae and others. The antibacterial component present in the clove was found to be heat stable as the autoclaving of the solutions did not affect its activity. Aqueous extracts of Rosa centrifolia (rose) and C. fistula (Amaltas) commonly used as therapeutic agents showed anti-microbial activity M. lysoditicus , Staph. aureus, C. pseudodiptheroid and Sh. dysenters , S. typhi, E. coli and S. pyogenes at significantly higher concentrations. Four fractions of organic extracts i.e. PI =P2, 80MR, EA2 and ALCR from the fruit of C. fistula were also were found to inhibit MDR Salmonella typhi at a Minimal Inhibitory Concentration (MIC) range of 125-500 ).µg/ml.

Methanolic extract of leaf extract of Bombax ceiba was found to be effective against S. epidermidis, S. typhi, B. Subtilis. Shemamin-a novel pure compound isolated from the leaf extract of Bombax ceiba showed antimicrobial activity against Listeria, B. subtilis, Ps. aeroginosa, Sh dysenteri, C. pseudo diptheroid and E.cloacae. Third group of plant products including Persea americana mill (avocado) peel, pulp, Beta vulgaris (beet) root peel and pulp, Porphyra tenera (nori, Sea vegetable) and Solanum tuberosu (potato) Peel, sub-peel and core were screened against several pathogens.. All the plant solutions and extracts had MIC value >25mg/ml except for Methanol extract of Persea americana peel that had MIC value for Klebsiella 6.25mg/ml, Proteus 1.5mg/ml, Candida 1.5 mg/ml, Pseudomonas 1.5mg/ml, E coli type I 12.5mg/ml, Staph aureus 6.25mg/ml, Strep sanguis, Strep sobrinus and Ecoli >25mg/ml.

Formulation of synergistic compound

Since it takes a long time to introduce new anti- microbial compound, a short cut strategy is to prepare synergistic formulations. In this study, synergistic preparations were developed by combining fruit solution of C fistula that is an important medicine of Ayuverdic and traditional medicne with amoxicillin a broad-spectrum antibiotic that has lost its efficacy. The new formulation developed was named as Amoxy-cassia. It has already been patented by the Government of Pakistan (Patent serial number 137124). We preferred the use of water solution as both amoxicillin and Cfistula fruit have proven efficacy in oral dosage in aqueous form. Amoxy-cassia was formulated first by screening with disc diffusion method and further confirmed by the checkerboard titration method and time kill kinetics.

This formulation is found effective against MDR Salmonella typhi, MRSA and Ecoli. MDR S typhi causes Typhoid Fever that is endemic in developing countries like Pakistan whereas MRSA is a common cause of nosocomial infections in the hospitalized patients. It has become the threat to hospital personnel, as it mostly invades the patients with knee and hip implantation. Urinogenital infections are very common especially among the women. Another synergistic combination, which was developed during this study, was between Shamimin and Amoxicillin named as Shamoxcillin. This preparation is found to be effective against S. typhi and E.coli.

In- vitro anti-adhesion Activity of the Plant Products

Infectious diseases can also be controlled by the inhibition of the attachment of bacterial strains to the host tissue. Because once pathogens are able to attain foothold on to the eukaryotic cell with the help of adhesins present in their pili, they survive there by enveloping themselves with the capsule that is recognized with difficulty by the human immune system. They then produce toxins and harmful products, which damage the tissues or interfere with the metabolic processes.

Most important step in the pathogenesis of the disease is the adhesion of the pathogens to the host surface through the adhesin present in their pili. Periodantal diseases and dental caries are due to the adhesion of bacteria to the salivary pellicle like S.sanguinis, Aggregation to the polymer dextran e.g. S sobrinus and coaggregation of A .naeshlundii to S sanguinis. They may enhance subsequent colonization of Porphyra gingivalis that is associated with adult periodontis. Organism was treated with the extracts for one hour. Three models were set up for the study of adhesion. First one was attachment of S.sanguis to human €˜O€™ group blood, second was aggregation of S sobrinus with dextran and third was coaggregation of A.naeslundii Tl4Vl with S.sanguis 35. Importance of controlling the dental plaque lies in the fact that most of the pathogens like Streptococcal species present in the dental plaque is responsible for causing pulmonary disease and bacterial endocarditis. The pathogens invade the lung and heart through oral cavity.

All four plants studied including - Beta vulgaris (beet) root, Persea americana mill (avocado), Solanum tuberosum (potato) and Porphyra tenera (sea vegetable -Nori) has the potential of interfering with the adhesion of bacteria to host epithelial surfaces . The most significant feature of this finding is that the water solutions of all the plants are playing an important role in the anti-adhesion activity of these plants. From this we can say that when avocado, Beetroot, Potato and sea vegetable is eaten, the components of the plant material get in contact with the bacteria present in plaque and thereby result in inhibition of colonization of bacteria on the teeth surface. Urinary infections caused mainly by E.coli are among the most common infectious diseases. Most of the uropathogenic E.coli can express type P. fimbriae and I that contain adhesin, which recognizes cell receptors present on the host cells. In our assays, we found that water extracts of P. tenera (sea vegetable) and B. vulgarts (beet) root inhibited the adhesion of E. coli to guinea pig RBC thus inhibiting infection of oral surfaces. Most significant inhibition of adhesion to RBCs was observation with P. americana (avocado) extract It would suggest that just like cranberry juice, avocado juice can also be consumed to avoid urinary tract infections with E.coli Type-1 . The result of our study reveals that all the four plants studied Beta vulgaris (beet) root, Persea americana mill (avocado), Solanum tuberosum (potato) and Porphyra tenera (sea vegetable) has the potential to interfere with the adhesion of all the oral bacteria.

Those combinations in which treated S.sanguis and A.naeshlundii which showed lower haemagglutination titer as compared to the titer of untreated bacteria were further checked by the flow cytometery technique. The flow cytometery result confirmed our first result obtained. Polyphenol Oxidase (PPO) and asparaginase are the two plants enzymes that show anti-adhesion activity in vitro. These compound were tested for their in-vivo anti-adhesion activity against

S. agalactiae (GBS) that normally resides in the female vagina and cause neonatal meningitis. GBS infects neonates during their passage through the vagina birth canal. It is one of the leading causes of due to neonatal meningitis. From our rat infection model it was concluded that treatment of animals post infection with plant enzymes like polyphenol oxidase, inhibited adhesion of bacteria. Another enzyme Aparaginase helped in the adhesion of GBS to vagina of the female rats. Thus PPO -plant enzyme can be used instead of antibiotics to avoid GBS infection.

Immune system plays an important role in the control infectious diseases . Enhancement of immune system is another important mode of controlling the infectious disease. In this study of Amoxy cassia and C. fistula were found to be effective immunomodulator. In this study effect of Amoxy cassia, fruit solution of C. fisula amoxicillin on immune system specific hurmoral immune system of BALB/c mice employing sheep RBC as the antigen. Uncountable antibodies producing cell were formed in the spleens of the mice treated with Amoxy cassia similarly the haemagglutination titer was highest too C. fistula fruit solution also exhibited immune enhancing property. Thus novel synergistic preparation Amoxy cassia has posses dual activity antimicrobioal and immune enhancing to combat with the pathogens. Our newly formed synergistic compound-Amoxy-cassia as well as fruit extract of C fistula exhibited a very significant immunoenhancing effect in BALB/c mice. This was observed as high titer of anti-SRBC antibody as well as uncountable anti-SRBC antibody producing plasma cells in spleens of mice treated with Amoxy-cassia. Control mice treated with saline showed fewer plaques /spleen as compared to mice receiving only C. fistula or amoxicillin alone. This study shows that amoxy cassia and C. fistula to be an immuno enhancing compound. They have the potential to be an excellent drug for the patient having impaired immune system like AID patients, person suffering from malignanssies and immunocompromised host. It we look at the efficacy of the amoxy cassia, it in vitro inhibits the nosocomially acquired MRSA infection.

This is the first report about the immunomodulatory properties of the C. fistula fruit extract Thus the novel synergistic formulation Amoxy-cassia is not only inhibiting the growth of difficult to treat organism including MDR Salmonella typhi strains, MRSA and, E.coli but it also enhances host immune response to specific antigens. Amoxycassia has the potential of being developed into an effective therapeutic agent for treatment of bacterial infections as well as an effective immunoenhacing substance to provide protection against infections.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
480.49 KB
2 1 Introduction/ Literature Review 1
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  1.1 Infectious Disease And Antibiotic Resistance 1
  1.2 Antimicrobial Products Of Plant Origin 2
  1.2 Combination Therapy For Infectious Diseases 12
  1.3 Mechanisms Of Pathogenesis Of Bacteria 15
  1.4 Effect Of Plant Products On Humoral And Cell Mediated Immune Responses 26
3 2 Materials And Methods 35
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  2.1 Preparation Of Plant Extracts 35
  2.2 Isolation And Identification Of Bacterial Strains 36
  2.3 Determination Of Susceptibility Of Bacterial Isolates To Standard Antibiotics And Plant Products 37
  2.4 Screening Of Plant Products On Growth Kinetics Of Susceptible Bacteria 40
  2.5 Effect Of Plant Products On The Pathogenesis Of Cariogenic Bacteria 41
  2.6 Effect Of Plant Products On The Attachment Of Uropathogenic E. Coli Type 1 42
  2.7 Flow Cytometric Evaluation Of The Effect Of Plant Products On Cariogenic And Uropathogenic Bacteria 43
  2.8 Effect Of Plant Products On S. Agalactiae Vaginal Infections In Rats 45
  2.9 Toxicity Of Plant Products For Balb / C Mice 46
  2.10 Effect Of Plant Products On Host Immune Response 46
4 3 Results 50
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  3.1 Isolation And Identification Of Bacterial Strains And Their Susceptibilities Against Standard Antibiotics 50
  3.2 Antimicrobial Activity Of Plant Products 50
  3.3 Synergistic Activity Of Amoxy-Cassia 52
  3.4 Effect Of Plant Products On Growth Kinetics Of Bacteria 54
  3.5 Effect Of Plant Products On The Pathogenesis Of Cariogenic Bacteria 55
  3.6 Effect Of Plant Products On Uropathogenic E. Coli Type 1 58
  3.7 Flow Cytometric Evaluation Of Anti-Adhesion Properties Of Plant Products 59
  3.8 Effect Of Plant Products On S. Agalactiae Vaginal Infection In Rats 59
  3.9 Toxicity Of Plant Products For Balb /C Mice 60
  3.10 Immunomodulation Of Host Response By Plant Products 61
5 4 Discussion 63
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  4.1 Antimicrobial Properties Of Plant Products 65
  4.2 Amoxy-Cassia; A New Synergistic Formulation For Mdr Salmonella And Mrsa 70
  4.3 Immunomodulating Properties Of Amoxy-Cassia 74
  4.4 Anti-Infective Plant Products For Cariogenic And Uropathogenic Bacteria 76
6 5 Conclusions 91
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7 6 References 92
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